Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis System

The antiapoptotic protein Bcl-xL is a major regulator of cell death and survival, but many aspects of its functions remain elusive. It is mostly localized in the mitochondrial outer membrane (MOM) owing to its C-terminal hydrophobic α-helix. In order to gain further information about its membrane organization, we set up a model system combining cell-free protein synthesis and nanodisc insertion. We found that, contrary to its proapoptotic partner Bax, neosynthesized Bcl-xL was spontaneously inserted into nanodiscs. The deletion of the C-terminal α-helix of Bcl-xL prevented nanodisc insertion. We also found that nanodisc insertion protected Bcl-xL against the proteolysis of the 13 C-terminal residues that occurs during expression of Bcl-xL as a soluble protein in E. coli. Interestingly, we observed that Bcl-xL increased the insertion of Bax into nanodiscs, in a similar way to that which occurs in mitochondria. Cell-free synthesis in the presence of nanodiscs is, thus, a suitable model system to study the molecular aspects of the interaction between Bcl-xL and Bax during their membrane insertion.


Introduction
Apoptotic programmed cell death is controlled by proteins of the Bcl-2 family (Bcl-2s). These proteins are functionally divided into three subfamilies: antiapoptotic proteins (e.g., Bcl-2 and Bcl-xL), multidomain proapoptotic proteins (Bax, Bak, and Bok), and BH3only proteins (e.g., Bid, Bim, and Bad) acting as modulators of the two other subfamilies. Altogether, the main function of Bcl-2s is to regulate the permeability of the mitochondrial outer membrane (MOM) to mitochondrial apoptogenic factors (e.g., cytochrome c and Smac/Diablo) that are released during the early steps of apoptosis. Typically, multidomain proapoptotic proteins Bax and Bak form large pores in the MOM, after being activated by BH3-only proteins such as tBid and Bim. This activation is counteracted by antiapoptotic proteins Bcl-2 and Bcl-xL through their physical interactions with Bax and Bak. This physical interaction can be prevented by other BH3-only proteins such as Bad (see [1][2][3][4] for recent reviews on the function and regulation of Bcl-2s).
A remarkable feature of Bcl-2s is the structural homology that exists between pro-and antiapoptotic proteins. Beyond the sequence homology that characterizes this family in the "Bcl-2 homology domains (BH)", structural data have shown that the soluble conformations of anti-and proapoptotic proteins are very similar (see [5][6][7][8] for reviews). However, this similarity cannot be extended to their membrane conformation. Indeed, proapoptotic proteins Bax and Bak are known to form large oligomers [9][10][11][12] that define a pore large enough to allow the passage of apoptogenic factors having sizes between~3.2 and 4.5 nm [13,14].

MALDI-MS Analysis
For MALDI-MS analyses, Bcl-xL-containing nanodiscs were added as above to Ni-NTA Sepharose (Qiagen) and washed thrice with 10 mM Tris/HCl (pH 8.0) and 500 mM NaCl. Beads were then placed in IP columns (Pierce, Appleton, WI, USA) before adding 200 µL of the same buffer supplemented with 50 mM sodium cholate to solubilize nanodiscs. Eluted Bcl-xL was then dialyzed thrice against Tris/HCl 20 mM (pH 7.2), NaCl 20 mM, and DTT 5 mM (preventing dimer formation) to eliminate cholate, and then concentrated on Vivaspin (10 kDa cutoff, Sartorius).
Dialysis steps are critical to reduce the amount of cholate and NaCl that could later interfere with the co-crystallization of the sample with the matrix. Samples were mixed (1:1 v/v) with a matrix solution of sinapic acid (10 mg/mL) in 1:1 (v/v) acetonitrile/deionized water with 0.1% trifluoroacetic acid. Each mixture (2 µL thereof) was dropped on the MALDI target plate using the dried-droplet method. MALDI-MS analyses were carried out on a MALDI-TOF mass spectrometer ( The mass spectra were acquired by averaging 8000 shots. The spectra were processed (including background subtraction and smoothing) using FlexAnalysis (Bruker). Experimental masses are the averages of two independent measurements.

Neosynthesized Bcl-xL Is Spontaneously Inserted into Nanodiscs
It is widely acknowledged that Bcl-xL, whether at endogenous levels or overexpressed, is essentially localized in the mitochondrial outer membrane [39]. This is likely due to the presence of a stretch of positively charged residues (termed the "X domain") located immediately upstream the C-terminal hydrophobic helix, that may stabilize its insertion by interacting with negatively charged lipid polar heads. Indeed, suppressing these positive charges decreased the mitochondrial localization of Bcl-xL, while replacing the homologous sequence of Bcl-2 by the X domain of Bcl-xL increased Bcl-2 mitochondrial localization [39].
Like we previously did for its proapoptotic partner Bax, we set up cell-free protein synthesis of Bcl-xL, in the absence [35] or in the presence [34] of nanodiscs. After the synthesis, reaction mixes were centrifuged at 20,000× g to check for the presence of precipitated protein. Whether in the absence or in the presence of nanodiscs, the vast majority of Bcl-xL was found in the supernatant ( Figure 1A). This was different from Bax, which remained soluble in the absence of nanodiscs, but precipitated in their presence [34]. Cell-free synthesized Bcl-xL appeared as several bands. It is now well established that Bcl-xL is the target of nonenzymatic deamidation on residues N52 and N66 located in the unstructured loop between α1 and α2 helices [29,30]. Cell-free protein synthesis conditions are likely favorable for Bcl-xL deamidation [37], because of pH (8.0), temperature (28 • C), and duration (16 h).
Like we previously did for its proapoptotic partner Bax, we set up cell-free protein synthesis of Bcl-xL, in the absence [35] or in the presence [34] of nanodiscs. After the synthesis, reaction mixes were centrifuged at 20,000× g to check for the presence of precipitated protein. Whether in the absence or in the presence of nanodiscs, the vast majority of Bcl-xL was found in the supernatant ( Figure 1A). This was different from Bax, which remained soluble in the absence of nanodiscs, but precipitated in their presence [34]. Cell-free synthesized Bcl-xL appeared as several bands. It is now well established that Bcl-xL is the target of nonenzymatic deamidation on residues N52 and N66 located in the unstructured loop between α1 and α2 helices [29,30]. Cell-free protein synthesis conditions are likely favorable for Bcl-xL deamidation [37], because of pH (8.0), temperature (28 °C), and duration (16 h).

Figure 1.
Neosynthesized Bcl-xL is spontaneously inserted into nanodiscs. (A) Bcl-xL was synthesized in a cell-free protein synthesis system in the absence (−ND) or in the presence (+ND) of nanodiscs. After a 16 h synthesis, the reaction mix was centrifuged at 20,000× g to separate precipitated proteins (in the pellet, p) and soluble proteins and nanodiscs (in the supernatant, sn). Contrary to its proapoptotic partner Bax [34], the presence of nanodiscs did not induce any precipitation of Bcl-xL. The Western blot is representative of more than 10 independent experiments. (B) Supernatant from the reaction mix +ND was incubated with Ni-NTA agarose to bind His7-tagged MSP1 nanodiscs. After washing, soluble Bcl-xL was found in the unbound fraction (u). After nanodisc elution with imidazole, nanodisc-associated Bcl-xL was found in the bound fraction (b). The Western blot is representative of more than 10 independent experiments. (C) The truncated mutant Bcl-xLΔC(213) was produced as in (A), and the association to nanodiscs was measured as in (B). After cell-free synthesis, the protein was present in the supernatant. However, after Ni-NTA binding, it remained in the unbound fraction, showing that it was not associated to nanodiscs. The Western blot is representative of two independent experiments. Nanodiscs were then purified from the supernatant by affinity chromatography on Ni-NTA. The presence of Bcl-xL was then probed in the flowthrough and bound fractions. We found that Bcl-xL was largely present in the bound fraction, showing that the protein was physically associated with nanodiscs ( Figure 1B).
As a negative control, we performed the synthesis of a mutant having a truncation after residue 213, lacking the whole C-terminal α-helix. This deletion was previously shown to impair the mitochondrial localization of Bcl-xL in both mammalian cells [39] and following heterologous expression in yeast cells [26]. We observed that, in the presence of nanodiscs, the truncated mutant Bcl-xLΔC(213) was still recovered in the supernatant; however, contrary to the full-length protein, it was not bound to nanodiscs ( Figure 1C). This confirmed that the insertion of Bcl-xL into nanodiscs was dependent on its C-terminal α-helix, which is akin to its insertion into biological membranes [26,39]. Neosynthesized Bcl-xL is spontaneously inserted into nanodiscs. (A) Bcl-xL was synthesized in a cell-free protein synthesis system in the absence (−ND) or in the presence (+ND) of nanodiscs. After a 16 h synthesis, the reaction mix was centrifuged at 20,000× g to separate precipitated proteins (in the pellet, p) and soluble proteins and nanodiscs (in the supernatant, sn). Contrary to its proapoptotic partner Bax [34], the presence of nanodiscs did not induce any precipitation of Bcl-xL. The Western blot is representative of more than 10 independent experiments. (B) Supernatant from the reaction mix +ND was incubated with Ni-NTA agarose to bind His 7 -tagged MSP1 nanodiscs. After washing, soluble Bcl-xL was found in the unbound fraction (u). After nanodisc elution with imidazole, nanodisc-associated Bcl-xL was found in the bound fraction (b). The Western blot is representative of more than 10 independent experiments. (C) The truncated mutant Bcl-xL∆C(213) was produced as in (A), and the association to nanodiscs was measured as in (B). After cell-free synthesis, the protein was present in the supernatant. However, after Ni-NTA binding, it remained in the unbound fraction, showing that it was not associated to nanodiscs. The Western blot is representative of two independent experiments. Nanodiscs were then purified from the supernatant by affinity chromatography on Ni-NTA. The presence of Bcl-xL was then probed in the flowthrough and bound fractions. We found that Bcl-xL was largely present in the bound fraction, showing that the protein was physically associated with nanodiscs ( Figure 1B).
As a negative control, we performed the synthesis of a mutant having a truncation after residue 213, lacking the whole C-terminal α-helix. This deletion was previously shown to impair the mitochondrial localization of Bcl-xL in both mammalian cells [39] and following heterologous expression in yeast cells [26]. We observed that, in the presence of nanodiscs, the truncated mutant Bcl-xL∆C(213) was still recovered in the supernatant; however, contrary to the full-length protein, it was not bound to nanodiscs ( Figure 1C). This confirmed that the insertion of Bcl-xL into nanodiscs was dependent on its C-terminal α-helix, which is akin to its insertion into biological membranes [26,39].

Nanodisc-Inserted Bcl-xL Is Full-Length
When produced as a recombinant protein in E. coli, Bcl-xL was present under two forms. The full-length protein was found in inclusion bodies, from which it could be resolubilized via a treatment with chaotropic agents (urea or guanidinium chloride) and further purified and reconstituted into nanodiscs using a co-formation method [21]. Before this study, soluble Bcl-xL was usually isolated from the supernatant, and, until 2015, investigators were unaware that this soluble fraction actually displayed a truncation that removed the 13 C-terminal residues. This mistake was caused by the abnormal migration of Bcl-xL on Tris/glycine SDS-PAGE, making both full-length (233 residues) and truncated (218 residues) Bcl-xL display a similar apparent size. As stated by Yao et al., "the finding that soluble Bcl-xL produced in bacteria is cleaved at the C-terminus impacts the many biochemical studies performed with recombinant N-terminal-tagged protein, which was presumed to include all C-terminal residues. Thus, effects attributed to the C-terminal tail may need to be reinterpreted in light of C-terminal truncation" [21]. It should be noted that many biochemical studies have been conducted with recombinant Bcl-xL in which the whole C-terminal α-helix was deleted on purpose to facilitate its production as a soluble protein [19,20,[40][41][42][43][44][45][46][47][48]. However, several studies relied on the synthesis of recombinant full-length Bcl-xL purified from E. coli lysate-soluble fractions where the C-terminal was likely truncated after residue 218 [46,[49][50][51].
We then tested if cell-free synthesis of Bcl-xL would produce a full-length protein or a truncated form. Bcl-xL truncation by bacterial protease(s) after residue 218 removes two C-terminal positively charged residues (R 230 K 231 ), which is expected to change the pI of the protein. Both nanodisc-inserted and non-inserted Bcl-xL were analyzed by IEF, and compared to Bcl-xL produced in E. coli, recovered either from inclusion bodies (full-length Bcl-xL) or from the soluble fraction (truncated Bcl-xL). We found that nanodisc-inserted Bcl-xL had a similar pI to Bcl-xL present in inclusion bodies (red line), while non-inserted Bxl-xL had a similar pI to Bcl-xL present in E. coli soluble fraction (green line) ( Figure 2). This suggested that nanodisc-inserted Bcl-xL was actually full-length, while non-inserted Bcl-xL was truncated. We also noticed the presence of an additional form (blue line) having an intermediate pI between full-length and truncated Bcl-xL (after residue 218), which was present in the non-inserted fraction of cell-free synthesis, but not in bacterial lysates. The nature of this form is unknown, and investigations are underway to determine whether its is due to cleavage at a position other than 218, or to a post-translational modification, such as deamidation [29,30]. Interestingly, we observed that co-producing Bcl-xL with its proapoptotic partner Bax (in the absence of nanodiscs) did not protect Bcl-xL against cleavage, since the full-length form was absent, seemingly increasing the proportion of this unidentified intermediate form compared to the truncated form after residue 218 ( Figure 2).  [21,37]; Bcl-xL produced in cell-free system, inserted into nanodiscs (fraction "b" in Figure 1B); Bcl-xL co-produced in cell-free system with its partner Bax, without nanodiscs [34]; Bcl-xL produced in cell-free system, not inserted into nanodiscs (fraction "u" in Figure 1B); Bcl-xL in the soluble lysate from E. coli (classical method, before [21]). The blot is representative of two independent experiments. (B) Densitometry scans of the lanes shown in (A). The red line marks the position of full-length Bcl-xL found in E. coli inclusion bodies, which is also found in cell-free synthesized bodies in E. coli [21,37]; Bcl-xL produced in cell-free system, inserted into nanodiscs (fraction "b" in Figure 1B); Bcl-xL co-produced in cell-free system with its partner Bax, without nanodiscs [34]; Bcl-xL produced in cell-free system, not inserted into nanodiscs (fraction "u" in Figure 1B); Bcl-xL in the soluble lysate from E. coli (classical method, before [21]). The blot is representative of two independent experiments. (B) Densitometry scans of the lanes shown in (A). The red line marks the position of full-length Bcl-xL found in E. coli inclusion bodies, which is also found in cell-free synthesized nanodisc-inserted Bcl-xL; the green line marks the position of truncated (after M218) found in E. coli soluble fraction (Yao et al., 2016) and in non-inserted cell-free synthesized Bcl-xL. The blue mark corresponds to an unknown form, which might have been generated by truncation at another position or by a post-translational modification such as deamidation. This form was only found in non-inserted cell-free synthesized Bcl-xL, but not in E. coli or in nanodisc-inserted cell-free synthesized Bcl-xL.
To further confirm that nanodisc-inserted Bcl-xL was full-length, a MALDI-MS analysis was performed on the protein. Nanodiscs containing Bcl-xL were bound on His-Trap and then solubilized by adding 50 mM sodium cholate. The eluate contained Bcl-xL and was free of His 7 -MSP1E3D1 ( Figure 3A). It was dialyzed to eliminate cholate and concentrated. MALDI-MS analysis showed that the protein had a mass of 26,046 Da (±10 Da), close to the theoretical mass of 26,049 Da for the full-length protein ( Figure 3B). As a matter of comparison, the full-length protein purified from E. coli inclusion bodies was analyzed in parallel and showed two peaks: a major one with a mass of 25,906 Da (±10 Da) and a minor one with a mass of 26,095 (±10 Da). The mass difference between the mass measured for nanodisc-inserted Bcl-xL and the mass measured for the main peak of the full-length protein purified from E. coli corresponds to 140 Da which is consistent with the mass of the initial methionine. The minor peak observed in the protein purified from inclusion bodies might correspond to the full-length protein including the initial methionine modified by an acetylation (+42 Da).   From these data, we could conclude that Bcl-xL synthesized in cell-free assays and inserted into nanodiscs was full-length, without the truncation of the 13 C-terminal residues [21], which would have generated a protein having a lower mass of 24,811 Da. The large width of the peak and the presence of a shoulder at a lower mass suggest that this methionine was also cleaved in a significant proportion of the protein produced in the cell-free system.

Bcl-xL Stimulates the Membrane Insertion of Bax into Nanodiscs
We previously reported that the presence of nanodiscs during the cell-free synthesis of Bax induced its partial precipitation, and that the protein was not inserted into nanodiscs [34]. We then tested if the co-synthesis of Bax and Bcl-xL could help Bax insertion. As we previously reported [34], the co-expression of Bcl-xL prevented Bax precipitation in the presence of nanodiscs ( Figure 4A). However, Bcl-xL∆C did not, suggesting that Bcl-xL membrane insertion was needed. We then tested whether Bax and Bcl-xL were actually inserted into nanodiscs. Nanodiscs in the supernatant from the expression of Bax alone or from the co-expression of Bax and Bcl-xL were bound to His-Trap to measure the association of Bax. When expressed alone, Bax was not associated to nanodiscs, whereas, when co-expressed with Bcl-xL, the association of Bax to nanodiscs was significantly increased since about half of the protein was in the bound fraction ( Figure 4C,D). As when expressed alone (Figure 1), Bcl-xL was almost completely bound to nanodiscs ( Figure 4C).   Nanodiscs from the Bax/Bcl-xL co-expression were then treated with sodium carbonate to measure the insertion of both proteins. Both proteins remained associated to nanodiscs, before or after the treatment, showing that they were actually inserted into nanodiscs ( Figure 4E). This stimulation of Bax membrane insertion by Bcl-xL is in line with previous observations showing that the overexpression of Bcl-xL increased Bax localization and insertion into the MOM of yeast cells, mouse fetal liver cells FL5.12, and human colorectal cancer cells HCT-116 [26].

Discussion
We showed that Bcl-xL could be produced in a cell-free system in the presence of nanodiscs, under conditions where the protein was efficiently inserted (Figure 1). Once inserted, the protein was protected from the cleavage that occurs in E. coli (Figures 2 and 3). It may be noted that, in spite of the presence of protease inhibitors, the cleavage likely occurred in the lysate since the migration of non-inserted Bcl-xL on IEF was similar to the migration of the protein recovered from the soluble fraction of lyzed E. coli cells, and it was different from the migration of the nanodisc-inserted protein and the protein recovered from E. coli inclusion bodies ( Figure 2). Interestingly, the co-expression of Bcl-xL with Bax allowed the latter to be inserted into nanodiscs, while Bax was not inserted when expressed alone ( Figure 4). This parallels the effect of Bcl-xL overexpression on Bax mitochondrial insertion in yeast and mammalian cells [26].
Since their description in the early 2000s [52], nanodiscs have been widely used to reconstitute membrane proteins, mainly for structural studies. Contrary to detergents, they provide a true membrane environment, and they are far more stable and homogenous in size than liposomes (see [53,54] for reviews). Their size can be adjusted through the use of different MSP variants, whereby the His 7 tag facilitates nanodisc purification to homogeneity. However, contrary to liposomes, they do not delimitate two compartments and, thus, cannot be used for transport studies.
Nanodiscs have been already used for structural NMR studies on Bcl-xL [55,56]. In the first study [55], Bcl-xL was deprived of the α1-α2 loop (residues 45-84) to facilitate its purification. In the second study [56], the protein was actually full-length (including the C-terminal α-helix) since it was purified from inclusion bodies. However, the purification followed a denaturing protocol, using reverse-phase HPLC. In a more recent study, the same group produced full-length Bcl-xL as a C-terminal fusion with intein that was nondenaturing and kept the C-terminus of Bcl-xL intact [22], a method previously been used by others [57,58]. In all these studies, Bcl-xL was first purified, and then reconstituted in nanodiscs using a co-formation protocol. To our knowledge, our work is the first exam-ple of Bcl-xL being spontaneously inserted into nanodiscs, thus limiting conformational alterations that may occur during the purification process.
Bcl-xL's proapoptotic partner Bax has also been reconstituted in nanodiscs. A tBidactivated monomer (or dimer) was shown to generate a lipidic pore in nanodiscs [59,60]. We recently reconstituted a constitutively active mutant under an oligomeric form [34]. In both studies, Bax was purified before being reconstituted in nanodiscs via co-formation. In the present study, owing to the presence of Bcl-xL, Bax could be directly inserted into preformed nanodiscs, and likely displays a conformation that might be similar to its conformation in cancer cells, where Bcl-xL is overexpressed and both proteins are inserted together into the MOM, as an inactive heterodimer [26]. This setup will be further used to get more precise insight into which residues/domains of the proteins are involved in their interaction in membranes. It is known that, for example, Bcl-xL-G138 and Bax-G67 are involved in their interactions in solution since replacements by larger residues break their interaction [61,62]. Furthermore, the C-terminal residues of Bcl-xL are required for Bcl-xL-mediated Bax retrotranslocation [25], but not for Bcl-xL-mediated Bax/MOM translocation [26]. The combination of cell-free Bax/Bcl-xL co-synthesis in the presence of nanodiscs with site-directed mutagenesis on both proteins will allow a better understanding of how they interact during these processes of translocation, retrotranslocation, and membrane insertion.

Data Availability Statement:
The data presented in this study are available on request from the corresponding authors.

Conflicts of Interest:
The authors declare no conflict of interest.